Structural features of reverse transcriptase (RT) that influence fidelity of reverse transcription in vivo are not well defined. We performed in vivo studies to identify structural determinants of murine leukemia virus (MLV) RT that influence fidelity by analyzing the effects of mutations in the YXDD motif and the dNTP binding site on the accuracy of DNA synthesis, and identified substitutions that increased the retroviral mutation rate. We also identified Y586F as a mutation in the MLV RT ribonuclease H (RNase H) primer grip subdomain that increased the mutation rate approximately fivefold-to date, the largest reported increase in the in vivo retroviral mutation rate. The Y586F mutation increased the frequency of substitutions 17-fold within 18 nt of adenine-thymine tracts (A-tracts), which induce DNA bending. These results suggest that when wild-type RT encounters irregular template-primer conformations such as those induced by A-tracts, the Y586 residue and the RNase H primer grip domain facilitate a template-primer conformation that is necessary for maintaining high fidelity of DNA synthesis. To elucidate mechanisms of RT fidelity, we are attempting to identify and characterize structural determinants of MLV and HIV-1 RTs that affect the retroviral mutation rate. We are examining the role of the MLV and HIV-1 RNase H primer grip subdomains in fidelity of DNA synthesis through mutational analysis. We also plan to determine the role of template-primer structure in RT fidelity. Finally, we are testing the error catastrophe hypothesis by determining the effects of mutator RTs and mutagenic nucleoside analogs on replication and evolution of MLV and HIV-1. Corresponds to Pathak Project 2 in the May 2003 site visit report of the HIV Drug Resistance Program